The lengthy and complicated multidrug therapy currently available to treat active tuberculosis (TB) infection has contributed to medical non-adherence and the emerging problems of multidrug-resistant (MDR)- and extensively drug-resistant (XDR)-TB, which are particularly deadly in the setting of HIV co-infection. In addition, persons with HIV are at increased risk for relapse even upon successful completion of anti-TB treatment. The prolonged therapy required to eradicate TB infection is believed to reflect the ability of Mycobacterium tuberculosis (Mtb) to persist within the host in a non-replicating state characterized by antibiotic tolerance to bactericidal drugs, which predominantly target actively dividing tubercle bacilli. Hyperphosphorylated guanosine ((p)ppGpp), which is synthesized by the stringent response enzyme RelA, plays an important role in bacterial growth restriction and antibiotic tolerance. RelA is essential for long-term Mtb survival during various in vitro and in vivo stress conditions. However, whether RelA is required for Mtb persistence in the host lacking cell-mediated immunity represents an important knowledge gap. Recently, we found that relA-deficient Mtb exhibits continued replication and metabolism during stress conditions, ultimately leading to bacillary death, as well as enhanced susceptibility to isoniazid during nutrient starvation and in the lungs of chronically infected mice. In collaboration with GlaxoSmithKline's Tres Cantos Open Lab for Diseases of the Developing World (GSK-DDW), we have screened a library of over 2 million compounds in RelA-inhibition and whole-cell killing assays, and performed preliminary cytotoxicity studies. A total of 178 RelA inhibitor candidates, representing 18 unique scaffolds, were identified and 12 of 39 compounds tested so far exhibit RelA-specific activity and synergy with isoniazid against nutrient-starved Mtb. The central hypothesis of this proposal is that releasing the molecular brakes on Mtb growth and replication through inhibition of the stringent response enzyme RelA is a safe and effective strategy to prevent Mtb persistence, decrease drug tolerance, and enhance susceptibility to bactericidal drugs in the CD4 T-cell-deficient host. Specifically, we will screen hits using whole-cell nutrient starvation assays. Target-specific candidates fulfilling physicochemical and toxicity criteria will be prioritized for safety/PK studis in immune-competent C57BL/6J mice. Up to 3 favorable RelA inhibitor candidates will be tested in dose-ranging studies alone and in combination with the first-line regimen in this mouse model, which is the background strain for the Cd4tm1Mak mice, which will be used to validate RelA as a target for Mtb persisters in the CD4 T-cell-deficient host. Our novel shock and kill strategy is expected to shorten the duration of treatment for drug-susceptible and drug-resistant TB in the HIV-infected and HIV-uninfected host. In addition to improving medical adherence and reducing the potential for the development of drug resistance, an abbreviated drug regimen to treat active TB would be particularly useful in HIV co-infected patients, since drug- drug interactions and immune reconstitution may complicate the concurrent management of both infections.